Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells

We constructed a gene library from a murine cell line with amplified dihydrofolate reductase (dhfr) genes by inserting random segments of DNA into lambda Ch4A. From this library, the dhfr gene and 30 kilobase pairs of surrounding DNA were cloned, and the restriction map was determined. All of the coding regions were sequenced and show that the gene spans a total of 31 kilobase pairs and has five intervening sequences in the coding portion of the gene. In addition, two classes of variant dhfr genes were found in the amplified line, which were amplified and present at levels of 10 to 30% of the normal dhfr genes. Numerous repeated sequences were located throughout the gene region, some of which share homology with previously defied families of repeats.     

Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines.

Murine 3T6 selected in increasing concentrations of methotrexate were unstable with respect to dihydrofolate reductase overproduction and methotrexate resistance when they are cultured in the absence of methotrexate. An analysis of the karyotypes of these resistant cells revealed the presence of numerous double minute chromosomes. We observed essentially identical kinetics of loss of dihydrofolate reductase gene sequences in total deoxyribonucleic acid and in deoxyribonucleic acid from fractions enriched in double minute chromosomes and in the numbers of double minute chromosomes per cell during reversion to methotrexate sensitivity, and this suggested that unstably amplified gene sequences were localized on double minute chromosomes. This conclusion ws also supported by an analysis of cell populations sorted according to dihydrofolate reductase enzyme contents, in which relative gene amplification and double minute chromosome content were related proportionally.     

Establishment and characterization of two human cell lines with amplified dihydrofolate reductase genes

Two SV40-transformed human cell lines, GM637, derived from a normal human subject, and GM5849, derived from a patient with ataxia-telangiectasia (A-T), were grown in increasing concentrations of the cytotoxic agent methotrexate (MTX). The GM637 line was naturally more resistant to methotrexate than was GM5849 and, over a 5-month period, became resistant even to very high concentrations (up to 100 microM). The GM5849 line became resistant to 500 nM methotrexate during the same period. However, dot blot and Southern blot analyses showed that both cell lines had amplified their dihydrofolate reductase (dhfr) genes to about the same extent, approx. 50-fold. Using the GM5849 line with amplified dhfr, we attempted to determine if interruption of DNA synthesis by hydroxyurea would cause DNA to be replicated twice within a single cell cycle, as has been reported for Chinese hamster ovary cells. No evidence for such a phenomenon was obtained.     

De novo synthesis of NGF subunits in S-180 mouse sarcoma cell line

It is an accepted hypothesis that the nerve growth factor protein (NGF) plays an important role in the development of vertebrate sympathetic and sensory ganglia and has effects on some central neurons. The best known NGF species is that isolated from the mouse submaxillary gland, MSG-NGF. MSG-NGF can be isolated as a subunit containing protein, 7S-NGF, made up of three dissimilar subunits called alpha-, beta-, and gamma-NGF. Beta-NGF is the biologically active subunit and its synthesis in vivo and in vitro has been demonstrated. Less is known about the synthesis of the alpha- and gamma-NGF or the assembly of the subunits into the 7S complex. In order to develop a clonal model system for the study of NGF synthesis, processing and secretion, affinity chromatography techniques were applied to cell extracts of S180 mouse sarcoma, a cell line known to synthesize NGF. After incubating S180 cells in³⁵S-Methionine, cell extracts were exposed to antibody directed against alpha-NGF, gamma-NGF or beta-NGF covalently bound to Sepharose beads in order to elute and characterize the desired NGF subunits. Parallel experiments using immunoabsorbed [³⁵S]Methionine-beta-NGF were carried out in the presence or absence of excess NGF, in order to demonstrate the specificity of this procedure. Affinity chromatography with a substrate analogue to arginine ester bound to Sepharose beads was also used to isolate de novo synthesized gamma-NGF. We were able to show that the S180 line synthesized alpha-, beta-, and gamma-NGF indistiguishable from alpha-, beta-, and gamma-NGF isolated from mouse submaxillary gland in terms of antigenic and physicochemical properties, and biological and enzymatic activities. These results are consistent with the hypothesis that NGF is synthesized, assembled and secreted by a single cell type.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Localization of dihydrofolate reductase amplified genes in Chinese hamster cells resistant to methotrexate

New sublines of BFFR1 and BFFR3 cells were obtained as a result of prolonged cultivation of Chinese hamster cells of Blld-ii-FAF 28 line (clone 431) in the presence of increasing concentrations of methotrexate (MTX). The lines obtained were resistant to 200 and 300 mcM of MTX, respectively. Amplification of the gene for dihydrofolate reductase (DHFR), similar to normal DHFR gene in restriction patterns, was proved by blot-hybridization of the resistant cells' DNA with 32P-labeled plasmid DHFR-26. Correlation is shown between the extent of gene amplification and resistance of the cell lines. In situ hybridization of the metaphase chromosomes of resistant cells with 3H-DHFR-26 results in preferential binding of the label with the regions of marker chromosomes 2 and 5, containing long, so called differential staining regions which are known to be the places of localization of amplified genes.     

The replication timing of the amplified dihydrofolate reductase genes in the Chinese hamster ovary cell line CHOC 400

We have examined the timing of replication of the amplified dihydrofolate reductase genes in the methotrexate-resistant Chinese hamster ovary cell line CHOC 400 using two synchronization procedures. DNA replicated in the presence of 5-bromodeoxyuridine was collected from cells of various times during the DNA synthesis phase and the extent of replication for defined sequences was determined by Southern blotting analysis of CsCl density gradient fractions. We report that under these conditions the DHFR gene replicates throughout the course of S phase in a mode similar to the bulk of the replicated genomic DNA. This contrasts with previous data that shows the non-amplified DHFR gene replicates during the first quarter of S phase. Therefore, we conclude that gene amplification alters the replication timing of the DHFR gene in CHOC 400 cells.     

Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences.

A modular dihydrofolate reductase gene has been introduced into Chinese hamster ovary cells lacking dihydrofolate reductase. Clones capable of growth in the absence of added nucleosides contain one to five copies of the plasmid DNA integrated into the host genome. Upon stepwise selection to increasing methotrexate concentrations, cells are obtained which have amplified the transforming DNA over several hundredfold. A detailed analysis of the chromosomes in three clones indicated the appearance of cytologically distinct chromosomal regions containing the amplified plasmid DNA which differ in surrounding sequence composition, structure, and location. Two of the clones examined have extensive, homogeneously staining regions. The DNA in these homogeneously staining regions replicates in the early part of the S phase. The amplified plasmid DNA is found associated at or near the ends of chromosomes or on dicentric chromosomes. We propose that integration of DNA may disrupt telomeric structures and facilitate the formation of dicentric chromosomes, which may then undergo bridge breakage-fusion cycles. These phenomena are discussed in relation to DNA transfer experiments and modes of gene amplification and chromosome rearrangement.     

Loss and stabilization of aminopterin resistance in murine cell lines

Mouse L-cell lines (B-82, tk-) were obtained using the stepwise selection method, their aminopterin (AP) resistance being 10(3)-5 X 10(4) times higher than that of parental cells. This resistance increase results from dihydrofolate reductase (DHFR) gene amplification which was determined from the 15-120-fold rise of the enzyme activity and with the cytogenetical techniques. The development and loss of AP resistance have been studied and karyological analysis of the lines obtained carried out. Two types of karyological changes were found in stable DM and HSR cells which correspond to extrachromosomal and intrachromosomal forms of the amplified material organization. Localization of the DHFR gene in HSR was proved using the in situ hybridization technique. Extrachromosomal localization of the amplified genes in DM providing unstable AP resistance is dominant at the early stages of the development of resistance and for a long time. It was demonstrated that DM and HSR can exist in one cell during the prolonged period. DHFR gene copy number in such cells is regulated by a change in the DM number, whereas the HSR size and localization are highly stable. HSR covers 1.7-1.9% of the genome length and 38-40% of the marker chromosome length. The genes localized in HSR provide stable AP resistance. Evidence on some intermediate, relative stabilization of the resistance has been obtained. This stabilization is mediated by temporary integration of DHFR copies into other chromosomal sites, in addition to HSR.     

Purification of dihydrofolate reductase amplified in Escherichia coli K-12

The Escherichia coli strain carrying pTP 6-10 which was constructed in our previous work (Iwakura, M., et al. (1983) J. Biochem. 93, 927-930) produces more than 400-fold dihydrofolate reductase as compared with the strain without the plasmid. Dihydrofolate reductase was highly purified from the cell-free extract of the plasmid strain simply by two steps; ammonium sulfate fractionation and ion-exchange chromatography. By 10-fold purification, the enzyme was essentially homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The restriction map of pTP 6-10 was also determined and the plasmid was shown to have an Ava I, an EcoR I, a Pst I, a Pvu I, and a Pvu II site. Our results indicate that the plasmid strain is suitable as a source of the enzyme and that plasmid pTP 6-10 is promising as a versatile plasmid vector for efficiently yielding the product of the cloned gene.     

Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line.

During stepwise increases in the methotrexate concentration in culture medium, we selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). We studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.     

Expression and amplification of engineered mouse dihydrofolate reductase minigenes.

We constructed mouse dihydrofolate reductase (DHFR) minigenes (dhfr) that had 1.5 kilobases of 5' flanking sequences and contained either none or only one of the intervening sequences that are normally present in the coding region. They were greater than or equal to 3.2 kilobase long, about one-tenth the size of the corresponding chromosomal gene. Both of these minigenes complemented the DHFR deficiency in Chinese hamster ovary dhfr-1-cells at a high frequency after DNA-mediated gene transfer. The level of DHFR enzyme in various transfected clones varied over a 10-fold range but never was as high as in wild-type Chinese hamster ovary cells. In addition, the level of DHFR in primary transfectants did not vary directly with the copy number of the minigene, which ranged from fewer than five to several hundred per genome. The minigenes could be amplified to a level of over 2,000 copies per genome upon selection in methotrexate, a specific inhibitor of DHFR. In one case, the amplified minigenes were present in a tandem array; in two other cases, a rearranged minigene plasmid and its flanking chromosomal DNA sequence were amplified. Thus, the mouse dhfr minigenes could be transcribed, expressed, and amplified in Chinese hamster ovary cells, although the efficiency of expression was generally low. The key step in the construction of these minigenes was the generation in vivo of lambda phage recombinants by overlapping regions of homology between genomic and cDNA clones. The techniques used here for dhfr should be generally applicable to any gene, however large, and could be used to generate novel genes from members of multigene families.     

Coregulation of dihydrofolate reductase and thymidylate synthase in overproducer cell lines of wild carrot

Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities are associated with a 285,000 molecular weight enzyme complex in carrot (Daucus carota L.). Selection for methotrexate (MTX) resistance by stepwise increase of the concentration of MTX results in a high frequency adaptation to MTX with little or no significant increase in DHFR activity. However, when as a second step following MTX selection a specific inhibitor of TS, 5-fluoro-2-deoxyuridine was used, DHFR overproducer lines were obtained. The overproduction phenotype of the lines was almost completely lost after 8 weeks of growth in the absence of selection pressure. Although DHFR and TS are independent gene products, their activities increase in proportion (~20-fold) in the overproducer lines. This strongly suggests that DHFR and TS are not only functionally and physically linked in the same enzyme complex, but also are coregulated. These cell lines resemble the MTX-induced DHFR overproducer amplified cell lines of mammalian origin in their mode of selection, high frequency of appearance, elevated enzyme activity, and increased specific mRNA levels.     

Changes in fatty acyl chains of phospholipids induced by interferon in mouse sarcoma S-180 cells

S-180 mouse sarcoma cells exhibit antiviral and antiproliferative responses to mouse β interferon. The composition of cellular phospholipids was specifically altered as a result of treatment with interferon. The unsaturated fatty acid content of all of the major phospholipids was decreased, resulting in an increase in the relative proportions of the saturated acyl side chains. These changes can be prevented by anti-interferon antibody and they are not observed with human interferon.     

A study of chromosomal changes associated with amplified dihydrofolate reductase genes in rat hepatoma cells and their dedifferentiated variants

We have examined the karyological consequences of dihydrofolate reductase gene amplification in a series of six rat hepatoma cell lines, all derived from the same clone. Cells of three of these lines express a series of liver-specific functions whereas those of three others fail to express these functions. Cells of each line have been subjected to stepwise selection for methotrexate resistance and, in most cases, resistance is associated with a 40-50-fold amplification of sequences hybridizing to a dihydrofolate reductase cDNA probe. In one line no modified chromosome is observed, whereas in two others the amplified genes are associated with an expanded chromosomal region. R- banding analysis of these karyotypes showed that few changes have occurred. These observations apply to two of the well-differentiated lines, and to a variant able to revert to the differentiated state. In contrast, in the two stably dedifferentiated hepatoma cell lines, amplified dihydrofolate reductase genes are found on large chromosomes of variable size, on ring chromosomes, and on chromosomes containing terminal, median, or multiple centromeres. We conclude that the nature of the chromosomal changes associated with dihydrofolate reductase gene amplification are the result of differences in cell lines rather than in the protocols employed for selection.     

Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain.

In previous studies, we utilized a neutral/neutral two-dimensional (2-D) gel replicon mapping method to analyze the pattern of DNA synthesis in the amplified dihydrofolate reductase (DHFR) domain of CHOC 400 cells. Replication forks appeared to initiate at any of a large number of sites scattered throughout the 55 kb region lysing between the DHFR and 2BE2121 genes, and subsequently to move outward through the two genes. In the present study, we have analyzed this locus in detail by a complementary, neutral/alkaline 2-D gel technique that determines the direction in which replication forks move through a region of interest. In the early S period, forks are observed to travel in both directions through the intergenic region, but only outward through the DHFR gene. Surprisingly, however, replication forks also move in both directions through the 2BE2121 gene. Furthermore, in early S phase, small numbers of replication bubbles can be detected in the 2BE2121 gene on neutral/neutral 2-D gels. In contrast, replication bubbles have never been detected in the DHFR gene. Thus, replication initiates not only in the intergenic region, but also at a lower frequency in the 2BE2121 gene. We further show that only a small fraction of DHFR amplicons sustains an active initiation event, with the rest being replicated passively by forks from distant amplicons. These findings are discussed in light of other experimental approaches that suggest the presence of a much more narrowly circumscribed initiation zone within the intergenic region.     

Regression of S-180 sarcoma using a xenogeneic antiserum

Tumour angiogenesis factor (TAF) was extracted from S-180 sarcoma grown as a solid tumour in Swiss albino mice. Its angiogenic activity was detected using the mouse intradermal and the chick chorioallantois membrane assays. Similar extracts from normal tissues failed to induce neovascularisation. An antiserum raised in rabbit, against TAF purified from mouse mammary adenocarcinoma was shown to neutralise the biological activity of TAF from S-180 sarcoma and also caused tumour regression in the mice. A possible therapeutic approach to solid tumours is indicated, as also the preparation of an immunotoxin.     

Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells.

We isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase amplicon types from the methotrexate-resistant CHO cell line CHOC400. The type I amplicons are 260 kilobases long, are arranged in head-to-tail fashion, and represent 10 to 15% of the amplicons in the CHOC400 genome. The type II amplicons are 220 kilobases long, are arranged in head-to-head and tail-to-tail configurations, and constituted the majority of the remaining amplicons in CHOC400 cells. The type II amplicon sequences are represented entirely within the type I unit. These are the first complete amplicons to be cloned from a mammalian cell line.